🧬 Appendix A — Integrated Summary

This appendix brings together dauer signaling theory, metabolic regulation pathways, and experimental validation methods used in the project. It links molecular signaling → physiological states → lab readouts.

🔁 Genetic Pathway of Dauer Formation (Figure A.1)

Dauer formation in C. elegans is controlled by three major genetic signaling branches, many involving daf genes (dauer formation genes).

🔀 The Three Branches

1. daf-1 (TGF-β branch)
   • Dauer-inhibiting pathway
   • Signals originate from ASI sensory neurons
   • Promotes reproductive growth under favorable conditions
2. daf-11 branch
   • Dauer-promoting pathway
   • Signals originate from ASJ neurons
   • Activated by stress cues (low food, high pheromone)
3. daf-2 (Insulin/IGF-1 signaling)
   • Inhibits dauer via phosphatidylinositol signaling
   • Integrates metabolic state with developmental fate

➡️ Figure logic:

• Arrows = activation
• T-bars = inhibition

Together, these branches decide whether the worm continues normal development or enters the stress-resistant dauer larval state.

🧪 A.0.1 Transforming Growth Factor-β (TGF-β) Pathway

🌱 Core Concept

The TGF-β pathway is a highly conserved signaling system controlling:

• Development
• Cell differentiation
• Cell-to-cell communication

While humans have 30+ ligands, C. elegans has only five, making it an elegant model.

🧬 TGF-β Ligands in C. elegans

• dbl-1
• daf-7
• unc-129
• tig-2
• tig-3

⚙️ Signaling Mechanism

1. Ligand binds type I (daf-1) and type II (daf-4) receptors
2. Receptors phosphorylate Smad proteins
3. Smads move into the nucleus → regulate gene expression

(unc-129 is the exception and uses a different receptor system)

🧠 daf-7: The Dauer Gatekeeper

• Expressed in ciliated sensory neurons
• Responds to:
  • Food availability 🍽️
  • Pheromones 🧪
  • Infection ⚠️
• Ortholog of human GDF-11 (linked to aging and nervous system development)

When daf-7 is ON:

• Smads daf-8 / daf-14 are activated
• Dauer-promoting factors daf-3 / daf-5 are suppressed
• ❌ Dauer entry prevented

When food is scarce:

• daf-3 / daf-5 become active
• ✅ Dauer formation occurs

Other Ligands

• dbl-1 → body size regulation (Sma/Mab pathway)
• unc-129 → axon guidance & cell migration (via netrin receptor unc-5)
• tig-2 / tig-3 → no confirmed biological roles yet

🍽️ A.0.2 Target of Rapamycin (TOR / mTOR)

🔋 What TOR Does

TOR is a nutrient-sensing kinase that regulates:

• Cell growth
• Metabolism
• Development
• Behavioral adaptation

🧩 TOR Complexes

• TORC1
• TORC2 (illustrated in Figure A.2)

⚠️ Unlike mammals, the connection between insulin signaling and TOR in C. elegans is not fully resolved.

❄️ TOR and Hypometabolism

TOR plays a crucial role in natural low-metabolism states, including:

• Dauer diapause (C. elegans)
• Hibernation 🐻
• Aestivation
• Hypoxia/anoxia tolerance

📌 Key insight: Dauer diapause is metabolically comparable to bear hibernation, highlighting deep evolutionary conservation.

🧪 A.0.3 Steroid Hormone Pathway

🧠 Key Genes

• daf-9 → steroid biosynthesis enzyme (cytochrome P450)
• daf-12 → nuclear hormone receptor (vitamin D receptor ortholog)

🧬 Dafachronic Acids

daf-9 converts cholesterol into:

• Δ4-dafachronic acid
• Δ7-dafachronic acid

These bile-acid-like steroids:

• Bind DAF-12
• Actively inhibit dauer arrest

🔄 Regulatory Features

• Cholesterol deprivation mimics daf-9 mutants
• This pathway acts downstream of many other daf genes
• Regulation differs from TGF-β and insulin signaling, despite functional overlap

📊 A.0.4 NanoDrop UV-Vis Spectroscopy (Figure A.3)

UV-Vis spectra (220–750 nm) were recorded for:

• SBS 1:10
• WBS 1:10
• WBS blue 1:10

Purpose:

• Assess protein concentration
• Detect sample purity
• Identify absorbance peaks indicating protein content

🧫 A.0.5 SDS-PAGE

• Used to separate serum proteins by size
• Pierce™ Prestained Protein Ladder used as molecular weight reference
• Applied to seasonal brown bear serum samples

🧬 A.0.6 Western Blot for Actin Quantification

Controls & Targets

• β-Actin → primary protein
• α-Tubulin → loading control

Key Observations (Figure A.6)

• Bands detected at:
  • ~30 kDa
  • ~80 kDa
• Chemiluminescence used for detection
• Unstripped actin bands visible beneath tubulin lanes

Ensures:

• Equal protein loading
• Reliable actin quantification

💪 A.0.7 Actin Staining for Muscle Fiber Width

• Fluorescent actin staining used to:
  • Visualize muscle fibers
  • Quantify muscle fiber width
• Enables assessment of muscle preservation or atrophy

🧠 A.0.8 ImageJ Macro Script for Mitochondrial GFP Analysis

What the Script Does (Figure A.7)

• Automates mitochondrial analysis:
  • Thresholding
  • Skeletonization
  • Branch density calculation
  • Shape metrics (e.g. eccentricity)

📌 Ensures:

• Reproducibility
• Objective image analysis
• Reduced user bias

📈 A.0.9 Normality Testing (Shapiro–Wilk)

Normality was tested for all quantitative datasets prior to statistical analysis.

Key Takeaways (Table A.1)

• Most groups showed normal distributions
• Some exceptions (p < 0.05), notably:
  • WBS 1:10 in multiple assays
  • Certain SBS Pure measurements

➡️ This justified:

• Use of parametric tests where appropriate
• Careful interpretation for non-normal groups

🧠 Big Picture Summary 🌍

This appendix tightly connects:

• Molecular signaling pathways (TGF-β, TOR, steroid hormones)
• Physiological adaptations (dauer, hypometabolism, muscle preservation)
• Experimental validation (NanoDrop, SDS-PAGE, Western blot, imaging, statistics)

Together, it provides the mechanistic backbone and methodological transparency for the entire study.